The present invention belongs to the field of protein purification, and more particularly, the invention relates to a method for purifying a recombinant protein.
Ion exchange chromatography is a chromatographic technique that is commonly used for the purification of proteins. In ion exchange chromatography, if the ionic strength of the surrounding buffer is sufficiently low, charged patches on the surface of the solute are attracted by opposite charges attached to a chromatography matrix. Elution is generally achieved by increasing the ionic strength (i.e. conductivity) of the buffer to compete with the solute for the charged sites of the ion exchange matrix. Changing the pH and thereby altering the charge of the solute is another way to is achieve elution of the solute. The change in conductivity or pH may be gradual (gradient elution) or stepwise (step elution). In the past, these changes have been progressive, i.e., the pH or conductivity is increased or decreased in a single direction.
At present, cation chromatography has been used for the purification of proteins in many methods. For example, Chinese patent No. 200410068790.3 discloses that cation exchange chromatography is used for removing acidic contaminants, which comprises increasing the relative conductivity to remove the acidic contaminants, decreasing the conductivity to equilibrate, and then increasing the conductivity to elute. The pH remains constant during the process. The purification results show that the acidic variant content is decreased by about 50%, and basic variants are not mentioned. Chinese patent No. 200880119331.X discloses that cation exchange chromatography is used, which comprises increasing the pH to wash, then decreasing the pH and increasing the conductivity to remove CHOP (Chinese hamster ovary protein), split protein A, DNA, aggregates and so on in the antibody, but does not involves acidic and basic-related proteins in the antibody.
Though the above methods have achieved the purpose of purifying the proteins partially, there still exist some problems of low removal rate of acidic and basic-related proteins and high loss rate of target protein.